Peroxynitrite (ONOO−/ONOOH) is a highly reactive biological oxidant formed under pathophysiologic conditions from the reaction of nitric oxide and superoxide radical anion. Peroxynitrite decomposes rapidly under physiological conditions to oxidizing intermediates, such as singlet oxygen (1O2), .NO2 free radical, and .OH free radical, that can damage biological targets. Pathological consequences associated with damage to biological targets can include oxidizing or nitrating of proteins, lipids, or DNA. Peroxynitrite crosses lipid membranes at a rate significantly faster than the rates of other known oxidants, indicating that this oxidant can travel distances of cellular dimensions. Thus, even in the presence of biological membranes, peroxynitrite can have free access to cellular interiors. Peroxynitrite is also known to nitrate tyrosine residues in proteins, and to oxidize sulfhydryls, methionines and macromolecules such as, for example, metalloenzymes, DNA, and lipids.
Peroxynitrite has been implicated in a variety of diseases and conditions. These diseases and conditions include, e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, dementia, atherosclerosis, post-infarction myocardial complications, arrhythmias, stroke, post-stroke tissue damage, acute respiratory disease syndrome, chronic obstructive pulmonary disease, pulmonary hypertension, vascular hypertension, cancer, ischemia-reperfusion injury, arthritis, inflammatory bowel disease, ulcerative colitis, AIDS, hepatitis, septic shock, chronic rejection of renal grafts, diabetes, gout, pancreatitis, meningitis, influenza, lupus, and radiation induced gastrointestinal injury.
Despite the relevance of peroxynitrite in biological systems and in the pathophysiology of several diseases, the study of this oxidant has been hindered by a lack of reliable and specific detection methods. Examples of known methods for detecting peroxynitrite include a method of detecting 8-nitroguanine produced by nitration of guanine, or nitrotyrosine produced by nitration of tyrosine, and a method of detecting singlet oxygen produced by reaction of peroxynitrite and hydrogen peroxide (H2O2) on the basis of light emission at 1.3 μm (Di Mascio et al., Methods Enzymol. 1996, 269:395-400). (Note that peroxide anion, OOH−, is likely the true reactive species in the case of hydrogen peroxide.) Although the first method achieves high specificity and has been widely used, that method requires the use of HPLC or immunostaining using an antibody, and therefore peroxynitrite cannot be detected in real time. In addition to the aforementioned two methods, there is a chemiluminescence method using luminol, and a fluorometric detection method using a 2′,7′-dichlorodihydrofluorescein (DCFH) fluorescence probe to detect overall active oxygen species (Wardman, Free Radic. Biol. Med. 2007, 43(7):995-1022). These methods, however, fail to achieve specificity for peroxynitrite, and therefore reliable detection cannot be expected even if various inhibitors are used. For example, in the method using 2′,7′-dichlorodihydrofluorescein (DCFH), DCFH reacts with both of nitrogen monoxide and superoxide to give an increase in fluorescence, and therefore it is impossible to distinguish whether peroxynitrite, nitrogen monoxide, or superoxide is detected.
Other chemical probes are known, but also suffer from a lack of specificity. For example, arylated fluorescein derivatives are known to be useful as fluorescent probes which do not react with nitrogen monoxide and superoxide. However, these probes not only react with peroxynitrite, but also react with reactive oxygen species such as hypochlorite ion and hydroxyl radical, and thus are not capable of achieving specific detection solely of peroxynitrite.
Accordingly, improved compounds, compositions, and methods for detecting and measuring peroxynitrite are needed that, inter alia, could be used to highly selectively visualize peroxynitrite in a living cell or tissue. For example, there is an ongoing need for non-invasive early markers of radiation induced gastrointestinal (GI) injury.